Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

7

Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

Abstract

Genomic analysis of ovarian cancer cell lines has revealed a panel that best represents the most common ovarian cancer subtype, high-grade serous ovarian cancer (HGSOC). However, these HGSOC-like cell lines have not been extensively applied by ovarian cancer researchers to date, and the most commonly used cell lines in the ovarian cancer field do not genetically resemble the major clinical type of the disease. For the HGSOC-like lines to serve as suitable models, they need to be characterized for common functional assays. To achieve that objective, we systematically studied a panel of HGSOC cells CAOV3, COV362, Kuramochi, OVCAR4, OVCAR5, OVCAR8, OVSAHO and SNU119 for migration, invasion, proliferation, clonogenicity, EMT phenotype and cisplatin resistance. They exhibited a range of efficacies and OVCAR5, OVCAR8 and Kuramochi were the most aggressive. SNU119 and OVSAHO cells demonstrated the lowest functional activities. Wide differences in expression of EMT markers were observed between cell lines. SNU119 were the most epithelial and OVCAR8 had the most mesenchymal phenotype. COV362 was the most resistant to cisplatin while CAOV3 was the most sensitive. Taken together, our systematic characterization represents a valuable resource to help guide the application of HGSOC cells by the cancer research community.

(A) Transwell migration assay was conducted using inserts with 8 μm pores and DMEM with 10% FBS as a chemoattractant. The number of migrated cells per field were imaged and counted (mean ± SD; 3 independent experiments). There were significant differences in the means across cell lines (p < 0.0001) as described in the results section. (B) Representative images of migrated cells for each cell line.

Evaluation of the ability of the panel of HGSOC cells to invade through matrigel

(A) Transwell invasion assay was conducted using growth factor reduced matrigel coated inserts with 8 μm pores. DMEM with 10% FBS served as a chemoattractant. The number of invaded cells per field were imaged and counted (mean ± SD; 3 independent experiments). There were significant differences in the means across cell lines (p < 0.0001) as described in the results section. (B) Representative images of invaded cells for each cell line.

The OC cells were seeded in 96-well plates (2000 cells/well) and allowed to grow for 4 days. Thereafter, their proliferation was measured using MTT assay and plotted to compare the growth rate of each cell line (mean ± SD; 3 independent experiments). There were significant differences in the means across cell lines (p < 0.0001) as described in the results section.

(A) The OC cells were seeded in 6-well plates (1000 cells/well) and allowed to grow to form visible colonies. Thereafter, the colonies were fixed, stained, imaged and counted to quantify the clonogenicity of these cell lines (mean ± SD; 3 independent experiments). There were significant differences in the means across cell lines (p < 0.0001) as described in the results section. (B) Representative images of colonies formed by each cell line.

Total RNA was isolated from the OC cells and used for reverse transcription followed by qRT-PCR using TaqMan gene expression assays (A) for vimentin and E-cadherin. Relative quantification was done using GAPDH as an internal control (mean ± SD; 3 independent experiments). (B) Western blot analysis of N-cadherin, vimentin, E-cadherin and claudin expression in the HGSOC cell lines using GAPDH as a loading control. Representative images of 3 independent experiments are shown.

The IC50 values for cispatin were determined in the cell lines by treating them with increasing doses of cisplatin for 24 h. This was followed by a 3-day recovery and then MTT assay was conducted to measure viability. IC50 was determined using Graph Pad (mean ± SD; 3 independent experiments).